Cysteinyl leukotrienes (cys-LTs) are lipid inflammatory mediators generated in vivo by mast cells (MCs), eosinophils, myeloid dendritic cells (DCs), basophils, and macrophages (reviewed in Kanaoka, Y., and J. A. Boyce. 2004. J. Immunol. 173:1503-1510). Cysteinyl leukotrienes abound in mucosal inflammation, play a validated role in human asthma (Wenzel, S. E., et al. 1990. Am. Rev. Respir. Dis. 142:112-119; Israel, E., et al. 1996. JAMA 275:931-936), and are important mediators in mouse models of pulmonary inflammation, remodeling, and fibrosis (Kim, D. C., et al. 2006. J. Immunol. 176:4440-4448; Beller, T. C., et al. 2004. J. Biol. Chem. 279:46129-46134; Henderson, W. R. Jr., et al. 2006. Am. J. Respir. Crit. Care Med. 173:718-728). Drugs that interfere with cys-LT synthesis (Israel, E., et al. 1996 JAMA 275:931-936) or that block the type 1 receptor for cys-LTs (CysLT1R) (Dahlen, S. E., et al. 2002. Am. J. Respir. Crit. Care Med. 165:9-14) are efficacious treatments for asthma, rhinitis, and nasal polyposis. Cys-LTs are synthesized from the precursor arachidonic acid following its liberation by calcium-dependent cytosolic phospholipase A2 (cPLA2) from membrane phospholipids (Clark, J. D., et al. 1991 Cell. 65:1043-1051) and its conversion to LTA4 by 5-lipoxygenase (5-LO) in concert with 5-lipoxygenase activating protein (FLAP) (Malavia, R., et al. 1993. J. Biol. Chem. 268:4939-4944; Dixon, R. A., et al. 1990 Nature 343:282-284). LTA4 is conjugated to reduced glutathione by LTC4 synthase (LTC4S), a homotrimeric integral nuclear membrane protein (Ago, H., et al. 2007 Nature 448:609-612). The cys-LTs comprise three distinct ligands. LTC4, the parent molecule, is exported to the extracellular space by a multidrug resistant protein after synthesis (Robbiani, D. F., et al. 2000 Cell. 103:757-768), where it is successively converted to LTD4 by γ-glutamyl leukotrienease-mediated removal of glutamic acid (Shi, Z. Z., et al. 2001. Molec. Cellul. Biol. 21:5389-5395). LTD4 is then converted to LTE4 by dipeptidase-mediated removal of glycine (Lee, C. W., et al. 1983. Immunology. 48:27-35). LTC4 is the only intracellular cys-LT, and LTD4 is the most powerful contractile agonist. The extracellular half-life of LTD4 is short (minutes) due to its rapid conversion to LTE4, effectively limiting its duration of action in vivo. LTE4 is stable and excreted in the urine (Sala, A., et al. 1990. J. Biol. Chem. 265:21771-21778). The stability of LTE4 accounts for the fact that it is the dominant cys-LT detected in biologic fluids. Consequently, LTE4 can be monitored in the urine (Drazen, J. M., et al. 1992. Am. Rev. Respir. Dis. 146:104-108), sputum (Lam, S., et al. 1988. J. Allergy Clin. Immunol. 81:711-717), and exhaled breath condensate (Csoma, Z., et al. 2002. Am. J. Respir. Crit. Care. 166:1345-1349) as an index of the cys-LT synthetic pathway activity in human disease states such as asthma.
To date, two G protein coupled receptors (GPCRs) for cys-LTs, respectively termed CysLT1R and the type 2 cys-LT receptor (CysLT2R) have been cloned and characterized (Lynch, K. R., et al. 1999. Nature. 399:789-793; Heise, C. E., et al. 2000. J. Biol. Chem. 275:30531-30536). These receptors share 38% amino acid identity. Each is 24-32% identical to the purinergic (P2Y) class of GPCRs that regulate cellular responses to extracellular nucleotides (Mellor, E. A., et al. 2001. Proc. Natl. Acad. Sci. USA. 98:7964-7969), suggesting a phylogenetic relationship between these two GPCR classes. The human CysLT1R, encoded by a gene on chromosome Xq21.13, is a high-affinity receptor for LTD4 (Kd˜1 nM) (Lynch, K. R., et al. 1999. Nature. 399:789-793), whereas the human CysLT2R is encoded by a gene on chromosome 13q14 and has equal affinity for LTC4 and LTD4 (Kd˜10 nM) (Heise, C. E., et al. 2000. J. Biol. Chem. 275:30531-30536). Although neither receptor has significant affinity for LTE4, the existence of an additional LTE4-reactive receptor has long been suspected. Early studies demonstrated that purified, synthetic LTE4 was more potent than LTC4 or LTD4 for inducing contraction of guinea pig tracheal rings (Lee, T. H., et al. 1984. Proc. Natl. Acad. Sci. USA. 81:4922-4925). Of the three cys-LTs, only LTE4 potentiated the contractile response of guinea pig trachea to histamine, a response that could be blocked by the administration of a nonselective inhibitor of the cyclooxygenase (COX) enzymes, indomethacin. LTE4 inhalation by asthmatic individuals potentiated their airway hyperresponsiveness (AHR) to subsequent challenges with either histamine or methacholine; this potentiation was blocked by oral administration of indomethacin (Christie, P. E., et al. 1992. Am. Rev. Respir. Dis. 146:1506-1510). Inhalation of LTE4, but not of LTD4, caused eosinophils, basophils, and MCs to accumulate in the bronchial mucosa of asthmatic individuals (Laitinen, L. A., et al. 1993. Lancet. 341:989; Gauvreau, G. M., et al. 2001. Am. J. Respir. Crit. Care Med. 164:1495-1500). Patients with exacerbated respiratory disease (AERD), a syndrome characterized by asthma, nasal polyposis, and marked cys-LT over-production, exhibit selectively enhanced bronchoconstriction in response to LTE4 relative to LTC4 or to histamine when compared to aspirin-intolerant asthmatic individuals (Christie, P. E., et al. 1993. Eur. Respir. J. 6:1468-1473). Thus, the potency of LTE4 as an inducer of inflammatory and physiologic effects in vivo is not explained by the pharmacology of the classical GPCRs for cys-LTs, which preferentially bind the metabolic precursors of LTE4. Thus, the three cys-LTs are all potent mediators, and show considerable tissue specificity for their respective actions. Both a 5-LO inhibitor (zileuton) and drugs that block CysLT1R (Knorr, B., et al. (1998) JAMA. 279:1181-1186) show clinical efficacy in asthma, despite the negligible activity of LTE4 at CysLT1R, and the fact that zileuton blocks only ˜50% of cys-LT generation in vivo (Israel, E., J. et al. (1996) JAMA. 275:931-936; Liu, M. C., et al., (1996) J. Allergy Clin. Immunol. 98:859-871). Identification of receptor(s) and pathways through which LTE4 exerts its effects may be highly significant in terms of the pathobiology of mucosal inflammation, as well as the treatment of asthma, AERD, and related diseases in which local concentrations of LTE4 are elevated and/or end-organ reactivity to LTE4 is high.
MCs are powerful effector cells relevant to asthma. They respond strongly to cys-LTs and are a useful cell type for modeling cys-LT-induced signaling events and receptor functions. It has been previously demonstrated that human and mouse MCs express both CysLT1R (Mellor, E. A., et al. 2001. Proc. Natl. Acad. Sci. USA. 98:7964-7969) and CysLT2R (Mellor, E. A., et al. 2003. Proc. Natl. Acad. Sci. USA. 100:11589-11593), and that these receptors constitutively form heterodimers on this cell type (Jiang, Y., et al. 2007. Blood. 110:3263-3270). Stimulation of MCs with LTD4, the most potent agonist of the CysLT1R, transactivates the Kit tyrosine kinase (Jiang, Y., et al. 2006. J. Immunol. 177:2755-2759), induces calcium flux (Mellor, E. A., et al. 2001. Proc. Natl. Acad. Sci. USA. 98:7964-7969), and phosphorylates mitogen activated protein kinase-kinase and its downstream effector, extracellular signal-regulated kinase (ERK) (Mellor, E. A., et al. 2002. J. Exp. Med. 195:583-592). These signaling events amplify MC proliferation (Jiang, Y., et al. 2006. J. Immunol. 177:2755-2759) and induce their generation of cytokines and chemokines (Mellor, E. A., et al. J. Exp. Med. 195:583-592). CysLT1R is required for all of these LTD4-induced responses, whereas CysLT2R acts to inhibit them (Jiang, Y., et al. 2007. Blood. 110:3263-3270). It was recently reported that LTE4 induces ERK activation and COX-2 expression, and causes prostaglandin D2 (PGD2) and macrophage inflammatory protein-1β (MIP-1β) generation by LAD2 cells, a well-differentiated human MC line (Paruchuri, S., et al. (2008) J. Biol. Chem. 283:16477-16487; Kirshenbaum, A. S., et al. (2003) Leukemia Res. 27:677-682), and to a lesser extent by primary cord blood-derived human MCs (hMCs). LTE4-mediated production of PGD2 by LAD2 cells was unaffected by short hairpin RNA (shRNA)-mediated knockdown of either CysLT1R or CysLT2R (Foster, C. J., et al. J. Clin. Invest. 107:1591-1598), supporting the presence of a previously unrecognized LTE4-reactive receptor on this cell type. ERK activation in response to LTE4, but not to LTD4, depended on indirect activation of the nuclear transcription factor peroxisome proliferator activated receptor (PPAR)-γ, which also was required for MIP-1β generation, COX-2 induction, and PGD2 generation. Moreover, LTE4-mediated production of PGD2 was unaffected by short hairpin RNA (shRNA)-mediated knockdown of either CysLT1R or CysLT2R, which respectively abrogated and amplified the responses to LTD4 stimulation (Paruchuri, S., et al. 2008. J. Biol. Chem. 283:16477-16487). These findings implied the existence of a distinct receptor-mediated pathway for the generation of inflammatory mediators in response to LTE4, occurring independently from the classical receptors.